The overall objective of this continuation of our ongoing project is to investigate the signaling and molecular mechanisms underlying the sustained infarct-sparing and anti-apoptotic effects observed during the late phase of ischemic preconditioning (PC). Although apoptosis has been shown to play a crucial role in cell death after ischemia/reperfusion injury, it remains largely unknown whether late PC protects against cardiac apoptosis and what signaling/molecular mechanisms underlie these protective actions. Building upon our previous works, we propose that brief episodes of mvocardial ischemia (ischemic PC protocol) protect against apoptotic cardiomyocyte death. Our fundamental hypothesis is that ischemic PC induces a sustained cardioprotected phenotype by activating pro-survival signaling pathways via the recruitment of stress-responsive transcription factors (STAT1 and STATS), leading to the upregulation of anti-apoptotic proteins that inhibit both the death receptor- and the mitochondria-dependent pathways of apoptosis. All studies will be conducted in an established murine model of ischemic PC. In Ajrn 1 we will determine whether ischemic PC inhibits cardiomyocyte apoptosis contributed to the delayed cardioprotective effects. Cardiac apoptosis will be evaluated by in situ hairpin-1-biotin ligation assay, DNA laddering, TUNEL assays, and immunohistochemistry of active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The influence of ischemic PC on the death receptor-mediated and the mitochondria-mediated pathways of apoptosis will be elucidated by examining their effects on the release of cytochrome c and Smac from mitochondria, on the activation of caspases (caspase-3, 8, and 9) and the cleavage of PARP, and on the expression of pro- and anti-apoptotic proteins. In Aims 2 and 3 we will establish whether the delayed anti- apoptotic actions afforded by late PC are mediated by STAT1 and/or STATS. The individual roles of STAT1 and STATS in mediating apoptotic cell death (the mitochondria-mediated vs. the death receptor-mediated apoptosis) will be conclusively established by targeted knockout of the STAT1 gene and by genetic inhibition of STATS via an inducible cardiac-specific STATS knockout mouse model. In Aim 4 we will identify whether the delayed cardioprotective effects conferred by late PC require both Tyr-705 and Ser-727 phosphorylation of STATS by using a Tyr-705 dominant negative mutant of STATS and a Ser-727 mutant of STAT3, respectively. This proposal will provide important new insights into the signaling and molecular mechanisms whereby the late phase of ischemic PC reprograms the heart in the intact animal.